RU2453599C1 - NUCLEIC ACID CODING FUNCTIONALLY ACTIVE RECOMBINANT IgAL PROTEASE NEISSERIA MENINGITIDIS OF SEROGROUP B, RECOMBINANT PLASMID DNA CONTAINING NUCLEOTIDE SEQUENCE CODING ACTIVE IgAL PROTEASE, PRODUCER STRAIN CONTAINING PLASMID DNA PRODUCING MATURE FORM OF IgAL PROTEASE, RECOMBINANT Ig PROTEASE NEISSERIA MENINGITIDIS OF SEROGROUP B, METHOD FOR PRODUCING MATURE FORM IgAL PROTEASE SHOWING IMMUNOGENIC AND PROTECTIVE PROPERTIES - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is described is nucleic acid coding IgAl protease Neisseria meningitidis of serogroup B, strain H44/76. What is presented is recombinant plasmid DNA containing nucleic acid. What is described is producer strain E.coli BL21 (DE3)/pBIGAPSl containing presented recombinant plasmid DNA. What is presented is a method for producing a mature form of IgAl protease Neisseria meningitidis of serogroup B. What is described is IgAl protease of molecular weight 107.6 kDa and estimated calculated isoelectric point 7.4.

EFFECT: invention allows producing high-purity IgAl protease as an ingredient of a polyvalent vaccine intended for protection of an individual, at first of children, against meningococcal infection and other microorganisms pathogenicity of which is caused by IgAl protease.

5 cl, 4 dwg, 5 ex

Description

The invention relates to the field of immunology, enzymology, microbiology and genetic engineering, and relates to a new nucleic acid encoding a functionally active recombinant IgA1 protease Neisseria meningitidis, a new recombinant plasmid DNA pBIGAPS1 and a new bacterial strain Escherichia coli BL21 (DE3) / pBIGAPS to obtain highly purified genetically engineered IgA1 protease and as a component of a multivalent vaccine designed to protect humans, especially children from meningococcal infections and other microorganisms pathogenicity due to IgA1 protease.

The problem of effective vaccine prophylaxis of meningococcal infection caused by meningococcus serogroup B has not yet been resolved. The WHO registry of registered meningococcal vaccines has not recorded a single drug against meningococcus of this serogroup. The lack of an effective meningococcal group B vaccine prevents the eradication of this infection in our country and beyond. It is known that serine IgA protease is secreted by some dangerous bacteria, primarily Neisseria meningitidis, and breaks down class A immunoglobulins on the surface of mucous membranes, destroying the first protective barrier of the host organism's immunity. Known meningococcal IgA1 protease, which in experiments on laboratory animals proved to be a highly effective meningococcal polio vaccine, and hence the vaccine against serogroup B meningococcus (application No. 2009136634/10, decision to grant a patent) dated August 16, 2010). It was found that IgA1 protease isolated from the culture fluid or from the purification products of the vaccine (meningococcal group A) is highly effective enzymatically and immunologically, but is contained in the indicated amounts in micro quantities, therefore, it may be difficult to obtain the protease in preparative amounts.

The prior art describes many attempts to create vaccines designed to protect against infection of group B meningitis. In the applications WO 2005/105141 20051110, WO 95/03413, WO 2005/102384 20051103, WO 03/094960 20031120, WO 2006/024954 20060309, WO 2004/014417 20040219, WO 2005/033148 20050414, WO 2004/032958, WO 03/028661 20030410, WO 2004/014419 A1 WO 02/083711 offer compositions and candidates for vaccines based on lipopolysaccharide, capsular oligosaccharides and a number of surface meningocidal proteins immunization of a patient against a disease caused by Neisseria meningitidis of various serogroups.

However, these compositions do not include IgA1 protease and do not provide sufficient effective protection against meningococcal infection and in particular from group B meningitis.

WO 2004/102199 A2 describes a method for identifying, selecting and isolating a vaccine component for passive and active immunization against a Streptococcus pneumonia microorganism that is destroyed by opsonophagocytic cells. The method of computer simulation (in silico) identified antigens inducing opsonophagocytosis, which are proposed to be used as components of a vaccine intended for immunization. Three pneumococcal streptococlipoproteinaromatase (SlrA) proteins, metalloprotease class IgA1 proteases and pneumococcal surface adhesion protein A (PsaA) from Streptococcus pneumonia were identified, and recombinant variants were obtained that were studied as vaccine components. It was found that the composition containing all three recombinant variants protects the mice from the specified infection, however, each component individually does not have protective properties.

Shishir K. Gupta et al., Vaccine, 2010, p. 28, pp. 7092-7097 describe the results of computer modeling of serogroup B meningococcal protein sequences and identified 6 epitopes of T-helper cell epitopes of three proteins: stimulating T cells protein A, motor protein A and IgA1 protease. The authors suggest that these peptides can be used as promising agents against group B meningococcus. However, experimental data confirming this hypothesis are not presented.

It is known (US Pat. US 7,235,242, Achtman, et al., 06/26/2007) to obtain an IgA1 protease fragment that is used as a carrier peptide. The synthesis of an IgA1 protease fragment containing from 40 to 200 amino acid residues and including 40 amino acid residues of the sequence SEQ ID NO: 1 is described using an automatic synthesizer. However, when conjugated to a polysaccharide, the IgA1 protease provides protection only against serogroup C meningococcus and is not effective against serogroup B meningococcus.

It is known (US Pat. US 7407653, Plaut et al., 08/05/2008) the use of bacterial IgA1 proteases for the treatment of diseases associated with IgA1 deposits in the tissues and organs of the body. Soluble IgA1 proteases containing various labels, for example, a polyhistidine fragment, are produced by a recombinant method. The enzyme is isolated by a known method using ultrafiltration, ion exchange and metal chelate chromatography. The resulting preparations can be used as an enzyme for the treatment of autoimmune diseases associated with the accumulation of IgA, but not as a vaccine.

Known closest to the claimed method for producing a precursor of IgA1 protease. The article S. Vitovski and JRSayers, Infection and Immunity, 2007, v. 75, No. 6, pp. 2875-2885 describes the cloning of a full-sized gene encoding a signal peptide, a secreted form of a proteinase and an autotransporter domain and the study of the mechanism of autoprocessing of IgA1 protease from it predecessor. For this purpose, a series of mutated forms of the serum group B Neisseria meningitidis serum B (NMB) precursor protein IgA1 were created, including a mutant with a deletion of a 32-membered peptide at the site of the autoprocessing site, as well as a mutant involving the replacement of the Ser residue in the active center with the Val residue. To obtain the mutants, NMB strains isolated from the cerebrospinal fluid of patients with meningococcus were used. It was found that these mutants did not possess autoproteolytic activity and the formation of secreted mature protease was not observed. But in this work, only the precursor of IgA1 protease was obtained, its protective and immunogenic properties were not investigated, and the possibility of creating a vaccine based on IgA1 protease was not considered.

In addition, analysis of the genomic data bank showed that to date, the sequence of the gene encoding the IgA1 protease from Neisseria meningitidis strain H44 / 76 is not known.

The invention solves the problem of producing a recombinant IgA1 protease having immunogenic and protective properties, intended to obtain a multivaccine for the prevention of meningococcal infections, including those caused by bacteria N.meningitidis serogroup B.

The problem is solved by:

Nucleic acid encoding IgA1 protease of Neisseria meningitidis serogroup B strain H44 / 76 (SEQ ID NO: 1);

recombinant plasmid DNA, comprising the above nucleotide sequence, and ensuring the expression of IgA1 protease in the host cell (pBIGAPS1), characterized by the following features:

chain length is 8123 bp .;

encodes a secreted form of IgA1 protease with a signal sequence at the N-terminus and a histidine tag at the C-terminus of the protein;

the nucleotide sequence encoding an IgA1 protease in the plasmid pBIGAPS1 is flanked by unique restriction sites by NdeI and XhoI endonucleases,

contains a T7lac transcription promoter;

the genetic marker is the nucleotide sequence (kan), which determines the resistance of bacterial cells transformed with plasmid pBIGAPS1 to kanamycin;

E. coli producer strain BL21 (DE3) / pBIGAPS1 containing recombinant plasmid DNA that produces a mature form of IgA1 protease;

IgA1 protease with a molecular weight of 107.6 kDa and a calculated isoelectric point of 7.4, with the amino acid sequence (SEQ ID NO: 2) encoded by the above nucleotide sequence, which has immunogenic and protective properties, designed to obtain a multivaccine for the prevention of meningococcal infections, including the number caused by bacteria N. meningitidis serogroup B;

and also due to the method of obtaining a mature form of IgA1 protease of Neisseria meningitidis serogroup B, which consists in the fact that the host cell is transformed with a recombinant plasmid DNA encoding IgA1 protease Neisseria meningitidis serogroup B with a nucleotide sequence (SEQ ID NO: 1), the producer strain is cultured, isolate and purify the target product from inclusion bodies.

The technical result of the invention is to obtain for the first time a highly purified full-length IgA1 protease of N. meningitidis serogroup B (40% of the enzyme from the total cellular protein, 20 mg from 2 g of cell biomass, the purity of the drug is not less than 90%). The resulting preparation has a pronounced immunogenic and protective activity in experiments on laboratory animals. The results of the work show the promise of the patented preparation of recombinant IgA1 protease in the development of a multivalent vaccine for the prevention of infections and, first of all, for the prevention of meningococcal infections caused by bacteria of N.meningitidis serogroup B.

One object of the present invention includes the establishment of the nucleotide sequence of iga Neisseria meningitidis serogroup B strain H44 / 76 encoding the IgA1 protease of N. meningitidis serogroup B. Another object of the present invention includes a recombinant plasmid comprising the above nucleotide sequence and allowing expression of the IgA1 protease in the host cell.

An analysis was made of the genomic data bank http://www.ncbi.nlm.nih.gov/Genbank) in order to search for known sequences of IgA1 protease genes produced by strains of N. meningitidis of serogroup B. It was revealed that the available data cover the only strain of serogroup B - MS58. Based on the sequence of the gene encoding IgA1 protease MC58, primers were designed to clone the gene fragment of the enzyme related strain H44 / 76 encoding the IgA1 protease form that is close in size to the secreted (~ 900-1000 a.a.). To clone the secreted form of the IgA1 protease enzyme, the nucleotide sequence was amplified according to the standard PCR protocol using the genomic DNA of the Neisseria meningitidis strain H44 / 76 as a template. A DNA fragment of about 2900 bp was obtained. The desired PCR product was cloned into pET-24a (+) vector (Novagen, Cat. No. 69749-3) using unique NdeI and XhoI restriction sites so that the protein produced contained His tag in the C-terminal region for subsequent purification protein using metal chelate chromatography. The cloned nucleotide sequence was sequenced; as a result, the nucleotide sequence of the gene fragment encoding the secreted form of the IgA1 protease from Neisseria meningitidis H44 / 76 was first established, based on which the amino acid sequence of the protein was determined. The resulting plasmid DNA was named pBIGAPS1.

Another object of the present invention includes an E. coli strain containing a recombinant nucleic sequence (see above) that produces a mature form of IgA1 protease. As the host cell, first of all, E. coli strain BL21 (DE3) was used, on the basis of which the strain BL21 (DE3) / pBIGAPS1 was obtained, which is described below.

Another object includes a method for producing a mature form of IgA1 protease of Neisseria meningitidis serogroup B, which comprises transforming the E. coli BL21 host cell (DE3) into the recombinant plasmid DNA encoding IgA1 Neisseria meningitidis serogroup B protease with the nucleotide sequence (SEQ ID NO: 1), host cells are cultured as described below, the target product is isolated and purified from inclusion bodies.

Another object of the present invention includes an IgA1 protease with a molecular weight of 107.6 kDa and an calculated isoelectric point of 7.4 and an amino acid sequence (SEQ ID NO: 2) encoded by the nucleotide sequence described above and obtained by the above method, designed to obtain a multivaccine for prevention infections, including those caused by bacteria N. meningitidis serogroup B.

IgA1 protease as an active ingredient can be used to obtain a multivaccine for the prophylaxis of infections caused by bacteria such as Clostridium ramosum, Bacteroides melaninogenicus, Capnocytophaga ochracea, Streptococcus pneumonia, Streptococcus sanguis, Streptococcus oralis, Streptococcus musemaeophus Haemophis Haemophis Haemophis Haemophis Haema , Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus viridans, Streptococcus canis, Streptococcus suis, Staphylococcus aureus, Staphylococcus epidermidis, Moraxella catarrhalis, Helicobacter pylbiorgeni Campylorium, Campyoris L. pylori, Campulus

To obtain the producer strain of IgA1 protease, competent cells of Escherichia coli BL21 (DE3) (E. coli B F ^- dcm ompT hsdS (r _B ^- m _B ^- ) gal λ (DE3)) (Stratagene Cat. No. 200131) of plasmid DNA are transformed pBIGAPS1.

The resulting strain, named E. coli BL21 (DE3) / pBIGAPS1, is characterized by the following properties:

Cultural and morphological signs:

The cells are small, rod-shaped, gram-negative, 1 × 3.5 μm, motile. The strain grows well on ordinary nutrient media (LB-broth, LB-agar). When growing on LB agar medium, the colonies are rounded, smooth, translucent, shiny, yellowish. The edge is even, the diameter of the colonies is 2-3 mm, the texture is pasty. Growth in a liquid LB medium is characterized by even turbidity, the sediment easily sedimentes.

Physiological and biochemical signs:

Cells grow at 4-42 ° C; optimum pH is 6.8-7.6. As a source of nitrogen, both ammonium mineral salts and organic compounds are used: amino acids, peptone, tryptone, yeast extract.

Genetic traits:

Cells show resistance to kanamycin (up to 100 μg / ml), due to the presence of the antibiotic resistance gene in recombinant plasmid DNA pBIGAPS1.

Storage conditions:

The bacterial strain E. coli BL21 (DE3) / pBIGAPS1 is stored on plates and jambs at 4 ° C. Reseeding on fresh media is carried out once a month. It can be stored for at least one year in LB medium containing 15% glycerol at -70 ° C.

Antibiotic resistance:

The cells of the producer strain show resistance to kanamycin (up to 100 μg / ml), due to the presence of the resistance gene in the recombinant plasmid DNA pBIGAPS1.

The invention is illustrated by graphic materials.

Figure 1. Physical map of recombinant plasmid DNA pBIGAPS1.

Recombinant plasmid DNA pBIGAPS1 containing 8123 bp encoding a secreted form of IgA1 proteinase, consisting of:

bigaps1 is the region encoding the signal peptide, the secreted form of the IgA1 protease and the histidine tag, and flanked by unique restriction sites by the endonucleases NdeI (3051) and XhoI (158) (the numbering is according to the numbering in the pET-24a vector), ori is the initiation site replication of recombinant plasmid DNA, T7lac promoter - hybrid transcription promoter, Stop - transcription terminator of the ribosomal operon E. coli, kan - gene that determines the resistance of the microorganism to kanamycin, f1 origin - region that allows to obtain single-stranded DNA, lacI - region, code uyuschaya lac repressor.

Figure 2. Enzymatic activity of IgA1 proteinase. The ordinate indicates the amount of non-hydrolyzed substrate — IgA1 or IgG (control by ELISA) after incubation with the enzyme.

Figure 3. The level of specific antibodies in the blood of immunized mice.

Figure 4. The protection of mice immunized with an IgA1 protease after infection with serogroup B meningococcus strain H44 / 76.

For the first time, the nucleotide sequence of the iga Neisseria meningitidis serogroup B nucleic acid (gene) fragment of strain H44 / 76, encoding the secreted form of an IgA1-specific protease, was established. For the first time, an expression strain of E. coli, a superproducer of serogroup B IgA1 protease, was created, a protein was isolated and refolded, allowing to obtain a soluble form of the enzyme.

The invention is illustrated by examples:

Example 1

Determination and preparation of nucleic acid encoding IgA1 protease of Neisseria meningitidis serogroup B strain H44 / 76 (SEQ ID NO: 1)

An analysis of the data of the genomic bank (http://www.ncbi.nlm.nih.gov/Genbank) revealed that so far the only genome of strain MC58 is known for Neisseria meningitidis serogroup B. Based on the assumption that the structure of IgA1 genes of proteases of related strains is similar, the primers were designed for cloning a fragment of a nucleic acid (gene) of strain H44 / 76 encoding a protease shape close to secreted (approximately 900-1000 amino acid residues A.O.). The selected structure of the primers allows amplification of the product of polymerase chain reaction (PCR), suitable for subsequent incorporation into expression vectors of the pET family (Novagen) used to obtain proteins in E. coli strains lysogenic by T7 bacteriophage.

For subsequent cloning in the pET-24 vector (a +) into primers (f1 — direct — 5-CGAGACAGCCATATGAAAACCAAACGTTTTAAAATTAAC-3 SEQ ID No. 3 — reverse — 5-GGGCTCGAGATTGTACAATCGGGGATATACCG-3 potential region SE44 ID4 / 76, the sequences of restriction enzyme recognition sites NdeI and XhoI (respectively for f1 and f2) introduced in the polylinker of the vector are introduced. The structure of primer f2, which does not contain termination codons, allows one to obtain a protein modified in the C-terminal region with a hexahistidine tag for subsequent metal chelate chromatography during isolation.

Amplification of a nucleic acid fragment is carried out according to the standard protocol for 25 cycles of PCR using the genomic DNA of strain H44 / 76 as a template. The annealing temperature of the primers is selected experimentally. Processing of the PCR fragment and vector with restriction enzymes, ligation and transformation of competent cells is carried out according to standard procedures. Next, the cloned DNA is sequenced from several primary clones, as a result, the nucleotide sequence of the nucleic acid fragment encoding the secreted form of the IgA1 protease from Neisseria meningitidis H44 / 76 was first established, based on which the amino acid sequence of the protein was determined (SEQ ID NO: 2). The resulting plasmid DNA was named pBIGAPS1.

Example 2

A) Obtaining recombinant plasmid DNA pBIGAPS1.

Recombinant plasmid DNA is obtained from overnight culture cells by the standard alkaline method (Maniatis, T., Fritsch, EF and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual, Cold Sping Harbor Laboratory Press) and used to introduce into competent cells using heat shock. For this, 100 ng - 1 μg of plasmid DNA is added to 50 μl of a suspension of competent cells and incubated for 40 minutes on ice. The resulting mixture was incubated at 42 ° C for 2 min, then incubated in ice for 2 min, 1 ml of cold LB broth was added and incubated for 1 h at 37 ° C. After this, 100 μl of a suspension of bacterial cells are plated on Petri dishes with agar containing 50 μg / ml kanamycin. The plates are incubated overnight at 37 ° C, the bacterial cells of several grown colonies are used for independent plating of microbiological tubes with 5 ml of LB liquid medium containing 50 μg / ml kanamycin, followed by growth of bacterial cells on a shaker at 37 ° C overnight. After that, glycerin is added to the tubes to a final concentration of 15%, the cell suspension is poured into plastic tubes and stored at -70 ° C.

A physical map of recombinant plasmid DNA is shown in figure 1.

B) Obtaining strain.

The E. coli BL21 (DE3) / pBIGAPS1 strain is obtained by introducing pBIGAPS1 plasmid DNA into competent E. coli BL21 (DE3) cells using the heat shock method. To obtain competent cells, the bacterial cells of the parent strain Escherichia coli BL21 (DE3) (E. coli B F ^- dcm ompT hsdS (r _B ^- m _B ^- ) gal λ (DE3)) are grown in LB liquid medium overnight at 37 ° C and used for seeding 100 ml of the same medium. The culture is grown to the early logarithmic phase on a rocking chair with intensive aeration (to optical density at 600 nm = 0.4 ÷ 0.5), it is quickly cooled in ice, the cells are precipitated by centrifugation for 10 min at 5000 rpm at + 4 ° С. The precipitate is washed twice in 50 ml of 100 mm CaCl ₂ . The precipitated cells are resuspended in 2 ml of 100 mM CaCl ₂ , with the addition of glycerol up to 10%. Divide into aliquots of 50 μl, quickly freeze and store at -70 ° C.

Example 3

Determination of the productivity of the producer strain of IgA1 protease.

In 15 ml of LB liquid medium containing 50 μg / ml kanamycin, a 1% inoculum of overnight cell culture was added and grown at 37 ° C on a shaker at 180 rpm for 2 hours until a turbidity of 0.8. The isopropylthiogalactopyranoside inductor (IPTG) is then added to a final concentration of 0.5 mM and incubation is continued for 2.0 hours under the same conditions. A 1 ml sample is taken and centrifuged for 6 minutes at 12,000 rpm, after which the cells are suspended in 100 μl of deionized water, add 33 μl of a buffer solution containing 125 mm Tris-HCl, pH 6.8, 20% glycerol, 3% sodium dodecyl sulfate, 3% mercaptoethanol and 0.01% bromophenol blue, heated for 10 min to 95 ° FROM. An aliquot was taken and analyzed by electrophoresis on a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate. The gel is stained with Coomassie R250 and scanned on a laser scanner. The productivity of E. coli strain BL21 (DE3) / pBIGAPS1 is 40% of the enzyme from the total cellular protein.

Example 4

Isolation of Recombinant IgA1 Protease

Cell disruption and lysate fractionation

Thawed cells after storage (2 g from 1 L of the induced culture) are resuspended in a seven-fold volume of 20 mM Tris-HCl buffer, pH 8.5, containing 1% Triton-X100, or in detergent-containing 20 mM potassium phosphate buffer, pH 7.5 Cell suspension five times sonicated and centrifuged (1 hour, 20,000 g). Separated fractions of soluble proteins (cell-free extract, BE) and the insoluble part of the cell lysate (debris and insoluble proteins) are isolated. According to the results of electrophoretic analysis, the target protein forms insoluble inclusion bodies (TB), regardless of the temperature conditions of the culture induction and the type of buffer used to destroy cells. The insoluble portion of the cell lysate containing TB, after resuspension in 1% Triton-X100 with repeated sonication and subsequent centrifugation, is used for chromatography on Q-Sepharose under denaturing conditions.

Anion exchange chromatography on Q-Sepharose

TB, isolated from 2 g of cells induced at 25 ° C, are dissolved in 20 ml of Tris-HCl buffer, pH 8.5, containing 6 M urea and 20 mm dithiothreitol (DTT). The total amount of protein contained in the TV is about 50 mg as determined by the Bradford method. After centrifugation and filtration, the solution was applied at a rate of 1 ml / min per 10 ml of anion exchange sorbent (two pre-packed HiTrap Q Sepharose HP cartridge columns, GE Healthcare, Sweden), equilibrated with the same buffer. Fractions containing purified protein and obtained as a result of gradient elution with sodium chloride (100 ml, 0-1 M NaCl) in the starting buffer are combined (20 ml in total) and used to convert the IgA1 protease into a soluble form. The yield of the partially purified target protein preparation at this stage is 40 mg from 2 g of cell biomass.

IgA1 protease refolding

The combined eluate (20 ml, 2 mg / ml) obtained after chromatography on Q-Sepharose was diluted 10 times with 20 mM Tris-HCl buffer, pH 8.5, by rapid dilution (digging out the protein solution into the buffer). After slowly mixing the solution (at a protein concentration of 0.2 mg / ml) for 20 hours and subsequent concentration by ultrafiltration to the initial volume, the protein solution is dialyzed three times against 1 L of 20 mM Tris-HCl buffer, pH 8.5. The solution is then centrifuged (30 min, 5000 g) and filtered through a sterile membrane with a pore size of 0.2 μm.

The final protein yield is 20 mg from 2 g of cell biomass. The purity of the drug is not less than 90%.

Example 5

Determination of immunogenic and protective activity

The immunogenic and protective activity of the obtained IgA1 protease was evaluated in Balb / C mice. To evaluate these parameters, animals are immunized with IgA1 protease twice intravenously at a dose of 20 μg / mouse with an interval of 45 days. For immunization of mice, a recombinant IgA1 protease preparation is used, prepared as described in Example 3. The resulting preparation has a specific enzymatic activity, i.e. hydrolyzes IgA1 and does not hydrolyze IgG (figure 2).

An indicator of the immunogenicity of the obtained IgA1 protease is the increase in specific antibodies in the blood of mice on the 10th day after immunization, determined by ELISA. 1 month after immunization, mice are infected with a live virulent culture of meningococcus serogroup B strain H44 / 47.

The protective activity of IgA1 proteases is estimated by the level of bacteremia in immunized animals 4 hours after infection with meningococcus, as well as by the number of surviving animals, on the 5th day after infection, compared with the same parameters in control (non-immunized) mice.

Figure 3 shows a diagram of the immunogenic activity of IgA1 protease. The ordinate shows the optical density of the sera at λ = 492 nm. After a single immunization, the level of specific antibodies in the sera of mice immunized with IgA1 protease increases by about 10 times. The antibody titer is 1: 1280-1: 2560. After re-immunization, antibodies to IgA1 protease were detected with a dilution of more than 1: 5000.

The diagram of figure 4 shows the level of bacteremia (in%) and the number of surviving mice, once immunized with IgA1 protease (shaded bar) compared to control, non-immunized animals (black bar). The bacteremia level in the group of immunized mice was reduced by 68% (32 CFU) compared with control non-immunized mice (100 CFU).

When mice were infected with meningococcus at a dose of 25 · 10 ⁴ microbial cells in the control group, only 2 animals out of 14 (14%) survived, while in the group immunized with IgA1 protease, 10 out of 14 mice survived (71%).

The results obtained indicate a high immunogenic and protective activity of the preparation of recombinant meningococcal IgA1 protease of serogroup B against this infection. In addition, the results obtained earlier (decision on the grant of a patent according to application No. 2009136634 dated October 5, 2009 for “A method for producing an IgA1 protease from a culture of Neisseria meningitidis serogroup A and an immunogenic preparation based on it”), suggest that the obtained preparation can be used as a candidate for a vaccine against meningococcal infection of the main epidemic serogroups A, B and C.

Thus, the nucleotide sequence of the iga Neisseria meningitidis serogroup B nucleic acid fragment of strain H44 / 76, encoding the secreted form of an IgA1-specific protease, was first established. For the first time, the expression strain E. coli superproducer of serogroup B IgA1 protease was created on the basis of the obtained plasmid DNA pBIGAPS1, the protein was isolated and refolded, allowing to obtain the IgA1 protease in an amount of 10-40 mg from 2 g of cell biomass.

The immunogenic and protective activity in experiments on laboratory animals and the potential of the patented preparation of recombinant IgA1 protease in the development of a multivalent vaccine for the prevention of infections and, first of all, for the prevention of meningococcal infections caused by bacteria of N. meningitidis serogroup B, as well as other pathogens whose activity due to the presence of IgA1 protease.

Claims (5)

1. Nucleic acid encoding IgA1 protease Neisseria meningitidis serogroup B strain H44 / 76 (SEQ ID NO: 1). 2. Recombinant plasmid DNA, comprising the nucleic acid according to claim 1 and providing expression of the IgA1 protease in the host cell (pBIGAPS1), characterized by the following features:
chain length is 8123 bp .;
encodes a secreted form of IgA1 protease with a signal sequence at the N-terminus and a histidine tag at the C-terminus of the protein;
the nucleotide sequence encoding an IgA1 protease in the plasmid pBIGAPS1 is flanked by unique restriction sites by NdeI and XhoI endonucleases,
contains a T7lac transcription promoter;
the genetic marker is the nucleotide sequence (kan),
determining the resistance of bacterial cells transformed with plasmid pBIGAPS1 to kanamycin. 3. The producer strain E. coli BL21 (DE3) / pBIGAPSl containing the recombinant plasmid DNA according to claim 2, which produces a mature form of IgA1 protease. 4. A method of obtaining a mature form of IgA1 protease of Neisseria meningitidis serogroup B, which consists in cultivating a producer strain according to claim 3, isolation and purification of the target product from inclusion bodies. 5. IgA1 protease with a molecular weight of 107.6 kDa and a calculated isoelectric point of 7.4, with the amino acid sequence (SEQ ID NO: 2) encoded by the nucleic acid according to claim 1, and obtained by the method according to claim 4, having immunogenic and protective properties designed to produce a multivaccine for the prevention of meningococcal infections, including those caused by bacteria N.meningitidis serogroup B.

Images